US20240012006A1 - A method of evaluating sperm health and identifying sperm to be used in assis reproductive technologies - Google Patents
A method of evaluating sperm health and identifying sperm to be used in assis reproductive technologies Download PDFInfo
- Publication number
- US20240012006A1 US20240012006A1 US18/044,511 US202118044511A US2024012006A1 US 20240012006 A1 US20240012006 A1 US 20240012006A1 US 202118044511 A US202118044511 A US 202118044511A US 2024012006 A1 US2024012006 A1 US 2024012006A1
- Authority
- US
- United States
- Prior art keywords
- sperm
- mrage
- health
- evaluating
- optionally
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 335
- 230000036541 health Effects 0.000 title claims abstract description 281
- 230000001850 reproductive effect Effects 0.000 title claims abstract description 57
- 238000005516 engineering process Methods 0.000 title claims abstract description 56
- 210000000582 semen Anatomy 0.000 claims abstract description 134
- 239000000523 sample Substances 0.000 claims abstract description 124
- 102000005622 Receptor for Advanced Glycation End Products Human genes 0.000 claims abstract description 64
- 108010045108 Receptor for Advanced Glycation End Products Proteins 0.000 claims abstract description 64
- 239000000090 biomarker Substances 0.000 claims abstract description 26
- 239000012528 membrane Substances 0.000 claims abstract description 22
- 239000012472 biological sample Substances 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 192
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 134
- 230000006907 apoptotic process Effects 0.000 claims description 49
- 238000000926 separation method Methods 0.000 claims description 47
- 230000019100 sperm motility Effects 0.000 claims description 42
- 230000035699 permeability Effects 0.000 claims description 35
- 208000000509 infertility Diseases 0.000 claims description 31
- 230000036512 infertility Effects 0.000 claims description 31
- 231100000535 infertility Toxicity 0.000 claims description 31
- 230000017074 necrotic cell death Effects 0.000 claims description 24
- 238000013467 fragmentation Methods 0.000 claims description 23
- 238000006062 fragmentation reaction Methods 0.000 claims description 23
- 238000012545 processing Methods 0.000 claims description 21
- 230000001640 apoptogenic effect Effects 0.000 claims description 18
- 206010012601 diabetes mellitus Diseases 0.000 claims description 17
- 230000002159 abnormal effect Effects 0.000 claims description 16
- 230000000391 smoking effect Effects 0.000 claims description 12
- 231100000527 sperm abnormality Toxicity 0.000 claims description 11
- 210000001700 mitochondrial membrane Anatomy 0.000 claims description 9
- 238000003744 In vitro fertilisation Methods 0.000 claims description 6
- 230000009027 insemination Effects 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 230000002596 correlated effect Effects 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 description 49
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 40
- 238000011156 evaluation Methods 0.000 description 31
- 238000000684 flow cytometry Methods 0.000 description 28
- 108020004414 DNA Proteins 0.000 description 26
- 108090000623 proteins and genes Proteins 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 23
- 230000004899 motility Effects 0.000 description 20
- 210000003934 vacuole Anatomy 0.000 description 16
- 108090000672 Annexin A5 Proteins 0.000 description 15
- 102000004121 Annexin A5 Human genes 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 15
- 238000003556 assay Methods 0.000 description 14
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 10
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- 238000001543 one-way ANOVA Methods 0.000 description 8
- 230000000750 progressive effect Effects 0.000 description 8
- 238000007427 paired t-test Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000003672 processing method Methods 0.000 description 6
- 239000012521 purified sample Substances 0.000 description 6
- 230000003068 static effect Effects 0.000 description 6
- 230000030833 cell death Effects 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 230000035558 fertility Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000001338 necrotic effect Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 4
- 208000007466 Male Infertility Diseases 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004807 localization Effects 0.000 description 4
- 230000004898 mitochondrial function Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011870 unpaired t-test Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 2
- 108050008874 Annexin Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 239000004429 Calibre Substances 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 241000282324 Felis Species 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108091093105 Nuclear DNA Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 231100000182 Sperm DNA damage Toxicity 0.000 description 2
- 238000012288 TUNEL assay Methods 0.000 description 2
- 241000282443 Ursidae Species 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 239000000104 diagnostic biomarker Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000007431 microscopic evaluation Methods 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000009612 semen analysis Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BMZRVOVNUMQTIN-UHFFFAOYSA-N Carbonyl Cyanide para-Trifluoromethoxyphenylhydrazone Chemical compound FC(F)(F)OC1=CC=C(NN=C(C#N)C#N)C=C1 BMZRVOVNUMQTIN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 108010079855 Peptide Aptamers Proteins 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000005787 mitochondrial ATP synthesis coupled electron transport Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000025608 mitochondrion localization Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000008789 oxidative DNA damage Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 229940080817 rotenone Drugs 0.000 description 1
- JUVIOZPCNVVQFO-UHFFFAOYSA-N rotenone Natural products O1C2=C3CC(C(C)=C)OC3=CC=C2C(=O)C2C1COC1=C2C=C(OC)C(OC)=C1 JUVIOZPCNVVQFO-UHFFFAOYSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 231100000521 sperm damage Toxicity 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 102000027257 transmembrane receptors Human genes 0.000 description 1
- 108091008578 transmembrane receptors Proteins 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 201000004822 varicocele Diseases 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
Definitions
- the present invention relates to a method of evaluating sperm health and identifying sperm to be used in Assisted Reproductive Technologies by providing a biological sample from a patient, preferably a semen sample; measuring the quantitative level of a biomarker in the sample; and evaluating sperm health based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- mRAGE membrane-bound receptor for advanced glycation end products
- the single greatest contributory factor to male infertility is sperm damage due to oxidative stress and resultant DNA fragmentation, estimated to account for between 30% and 80% of cases.
- DNA fragmentation affect sperm quality, it has also been found to negatively impact embryo quality, implantation rates and miscarriage rates.
- Tests include the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-nick-end labelling (or TUNEL), comet, or sperm chromatin dispersion (SCD) assays.
- TdT terminal deoxynucleotidyl transferase
- dUTP deoxyuridine triphosphate
- TUNEL blunt-end labelling
- comet or sperm chromatin dispersion
- Extracellular markers that correlate with the degree of sperm DNA damage and the quality of the sperm would be highly valuable for the identification and removal of damaged sperm prior to assisted reproductive techniques (ART).
- Significant efforts have been made over the last three decades to identify a more sophisticated marker.
- Mainly intracellular markers of DNA fragmentation, oxidative DNA damage, oxidative stress, mitochondrial function and sperm cell death have been identified. However, these require invasive internal labelling procedures leaving the sperm non-viable afterwards. In this study, we evaluate an extracellular marker of sperm cell health.
- the Receptor for Advanced Glycation End products is a central signalling molecule of the innate immune system and belongs to a class of cell-surface, pattern recognition receptors encoded at the MHC class III locus. It exists in numerous forms as membrane-bound (full length), soluble or secretory protein. Soluble RAGE (sRAGE) is produced by both proteolytic cleavage of membrane-bound/full length RAGE (mRAGE/fl-RAGE), and alternative mRNA splicing. The soluble isoforms include the extracellular domains but lack the transmembrane and cytoplasmic domains
- RAGE is long established as a key contributor to severe chronic pathologies including neurodegeneration, atherosclerosis, cardiovascular diseases, osteoarthritis and diabetes. Under normal conditions, the expression of RAGE is very low. However, during disease, the production of RAGE is highly induced on the cell surface by a number of different ligands such as advanced glycation end products (AGE), high mobility group box-1 (HMGB1), S100/calgranulins, phosphatidylserine, C3. In addition, RAGE expression is further enhanced after immune activation or infection and many RAGE ligands are secreted by monocytes, macrophages, neutrophils and leukocytes which are likely to be crucial for the initiation and propagation of a RAGE-dependent inflammatory response. The location of the membrane-bound receptor at the acrosomal cap of human sperm, which is a direct access point to nuclear DNA within the sperm head, has been implicated in playing a role in mediating and exacerbating nuclear DNA damage.
- mRAGE as a diagnostic biomarker of low-quality sperm.
- mRAGE expression was analysed on sperm from non-diabetic, non-obese and non-smoking men thereby removing the heightened effects of RAGE and its ligands on sperm health in these conditions.
- Flow cytometry was used to quantitatively assess mRAGE expression at a cellular level on sperm and computer-assisted sperm analysis (CASA) was used to evaluate sperm motility.
- CUA computer-assisted sperm analysis
- a method of evaluating sperm health comprising the steps of:
- the method of evaluating sperm health is an in vitro method.
- mRAGE is bound to a membrane of a cell.
- mRAGE is bound to the membrane of a sperm.
- mRAGE is bound to the membrane of the sperm head.
- mRAGE is bound to the membrane of the equatorial regions of the sperm head.
- mRAGE is a transmembrane receptor.
- mRAGE comprises three extracellular domains.
- mRAGE comprises three extracellular domains—a V-type Ig domain and two C-type Ig domains (C1 and C2).
- mRAGE comprises three extracellular domains—a V-type Ig domain and two C-type Ig domains (C1 and C2); and a transmembrane domain.
- mRAGE comprises three extracellular domains, a V-type Ig domain and two C-type Ig domains (C1 and C2); a transmembrane domain; and a cytoplasmic tail.
- mRAGE is full-length RAGE (fl-RAGE).
- the biological sample is a semen sample.
- the biomarker is a gene.
- the biomarker is a nucleic acid.
- the biomarker is a deoxyribonucleic acid.
- the biomarker is a ribonucleic acid.
- the biomarker is a protein.
- the biomarker is a peptide.
- the biomarker for evaluating sperm health is a gene having a GenBank Accession Version Number AQY76652
- the biomarker for evaluating sperm health is a protein having a UniProt ID Q15109
- the measuring step (b) comprises measuring the quantitative level of mRAGE on sperm cells from a semen sample.
- the measuring step (b) comprises measuring the quantitative level of mRAGE on sperm cells from a semen sample by any quantitative analytical technique.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample by a cell-based assay.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample by flow cytometry.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a RAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a sRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a mouse anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a rabbit anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a rat anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a sheep anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal rabbit anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal rat anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal sheep anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal mouse anti-human mRAGE antibody.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using the monoclonal mouse anti-human mRAGE antibody—AF647.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using DNA aptamers.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using RNA aptamers.
- the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using peptide aptamers.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the presence of mRAGE is defined as mRAGE positive.
- the evaluating step (c) comprises evaluating the presence or absence of quantifiable levels of mRAGE on sperm cells from a semen sample to define the patient as mRAGE negative or mRAGE positive.
- the presence of quantifiable levels of mRAGE on sperm cells from the semen sample defines the patient as mRAGE positive and wherein the absence of quantifiable levels of mRAGE on sperm cells from the semen sample defines the patient as mRAGE negative.
- the evaluating step (c) comprises comparing the amount of sperm expressing quantifiable levels of mRAGE to the amount of sperm not expressing quantifiable levels of mRAGE.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 1-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 2-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 3-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 4-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 5-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 6-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 7-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 8-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 9-fold.
- the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 10-fold.
- the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample.
- the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample to define the patient as mRAGE negative or mRAGE positive.
- the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE negative is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is lower than 10-fold compared to the mRAGE positive sperm cells.
- MFI Mean Fluorescent Intensity
- the evaluating step (c) comprises evaluating the percentage of sperm expressing qualifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 10-fold higher compared to the mRAGE negative sperm cells.
- MFI Mean Fluorescent Intensity
- the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 20-fold higher compared to the mRAGE negative sperm cells.
- MFI Mean Fluorescent Intensity
- the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 30-fold higher compared to the mRAGE negative sperm cells.
- MFI Mean Fluorescent Intensity
- a Mean Fluorescent Intensity value expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value not expressing quantifiable levels of mRAGE by less than 10-fold.
- a Mean Fluorescent Intensity value expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value not expressing quantifiable levels of mRAGE by at least 10-fold.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is indicative of poor sperm health.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE negative is indicative of healthy, fertile sperm.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of abnormal sperm.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of normal sperm.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of good sperm health.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of one or more of normal sperm and good sperm health.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of one or more of poor sperm health and abnormal sperm.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of poor sperm motility.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm and poor sperm motility.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of infertility.
- the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm, poor sperm motility and infertility.
- the patient is a mammal.
- the patient is non-human
- the patient is non-human, such as bovine, equine, canine, feline, porcine, ovine and ursidae animals.
- the patient is human.
- the patient is non-diabetic human.
- the patient is non-diabetic, non-smoking human.
- the patient is non-diabetic, non-smoking, non-obese human.
- the patient is non-diabetic, non-smoking, non-obese (BMI ⁇ 29.9) human.
- the patient is non-diabetic, non-smoking, non-obese (BMI ⁇ 29.9) man.
- the method of evaluating sperm health further comprises assessing sperm motility.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein the sperm are categorised as immotile (IM), non-progressively motile (NP) or progressively motile (PR).
- IM immotile
- NP non-progressively motile
- PR progressively motile
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health and poor sperm motility.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as NP move, but do not move in a substantially linear direction.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as NP move, but do not move in a substantially linear direction, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as PR move in a substantially linear direction.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as PR move in a substantially linear direction, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 10% of sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 20% of sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 30% of sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 32% of sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 32% of sperm categorised as PR in a semen sample is considered normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 10% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 20% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 30% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 40% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 40% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm motility, wherein the percentage of mRAGE positive sperm is inversely correlated to the percentage of PR sperm; defined as sperm with a space gain of ⁇ 5 ⁇ m s ⁇ 1 .
- the method of evaluating sperm health further comprises assessing sperm morphology.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein the sperm are classified as normal or abnormal.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein the sperm are classified as normal or abnormal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; and there is a well-defined acrosomal region comprising 40-70% of the sperm head area.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; and there is a well-defined acrosomal region comprising 40-70% of the sperm head area, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; and the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; and the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head, as defined by the World Health Organization (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; and the midpiece of the sperm is slender, regular and substantially the same length as the sperm head.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; and the midpiece of the sperm is slender, regular and substantially the same length as the sperm head, as by the defined World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; and the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; and the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head; and the principal piece has a uniform calibre along its length, is thinner than the midpiece, and is approximately 45 micrometres long.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head; and the principal piece has a uniform calibre along its length, is thinner than the midpiece, and is approximately 45 micrometres long, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, which is indicative of poor sperm health, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- WHO World Health Organization
- the method of evaluating sperm health further comprises a step of assessing mitochondrial membrane potential (MMP).
- MMP mitochondrial membrane potential
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP; as defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is 10-fold, or lower, than the mRAGE negative sperm cells, which is indicative of poor sperm health.
- MFI Mean Fluorescent Intensity
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros stains cells with high MMP.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros stains cells with high MMP, which is indicative of good sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with low MMP is present in mRAGE positive sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with low MMP is present in mRAGE positive sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm, which is indicative of good sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm, which is indicative of good sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm, which is indicative of good sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm, which is indicative of good sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to mRAGE negative sperm.
- quantifiable levels of MMP is indicative of mRAGE negative sperm and the absence of quantifiable levels of MMP is indicative of mRAGE positive sperm.
- the method of evaluating sperm health further comprises the step of assessing cell permeability.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, and positive staining by DAPI represents non-viable cells.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein at least a 2-fold increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein at least a 3-fold increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- At least a 2-fold decrease in non-permeable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health
- the method of evaluating sperm health further comprises the step of assessing apoptosis.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V and positive staining with Annexin V is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and positive staining with Annexin V in combination with DAPI is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 2-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is evaluated by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 4-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 4-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- the method of evaluating sperm health further comprises the step of assessing DNA fragmentation.
- the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay.
- TUNEL ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling
- the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein an increase in DNA fragmentation is present in mRAGE positive sperm, which is indicative of poor sperm health.
- TUNEL ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling
- the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein an increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- TUNEL ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling
- the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein at least a 2-fold increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- TUNEL ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling
- the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein at least a 3-fold increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- TUNEL ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling
- the method of evaluating sperm health further comprises the step of identifying sperm that can be used in assisted reproductive technologies (ART).
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including intrauterine insemination (IUI).
- ART assisted reproductive technologies
- IUI intrauterine insemination
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI) and intracytoplasmic injection (ICSI).
- ART assisted reproductive technologies
- IUI intrauterine insemination
- ICSI intracytoplasmic injection
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI), intracytoplasmic injection (ICSI) and in vitro fertilisation (IVF).
- ART assisted reproductive technologies
- IUI intrauterine insemination
- ICSI intracytoplasmic injection
- IVF in vitro fertilisation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI), intracytoplasmic injection (ICSI), in vitro fertilisation (IVF) and artificial insemination (AI).
- ART assisted reproductive technologies
- IUI intrauterine insemination
- ICSI intracytoplasmic injection
- IVF in vitro fertilisation
- AI artificial insemination
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including density gradient centrifugation (DGC).
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including the sperm swim-up method.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including the sperm swim-up method, as defined World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- ART assisted reproductive technologies
- WHO World Health Organization
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm; and the swim-up method relies on sperm motility.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm; and the swim-up method relies on sperm motility to separate PR sperm from IM sperm and NP sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in a decrease in mRAGE positive sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in a decrease in mRAGE positive sperm.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in mRAGE positive sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in mRAGE positive sperm, which is indicative of good sperm health.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm and the swim-up method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 2-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 3-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 5-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DCG method results in an increase in PR sperm.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim up method results in an increase in PR sperm.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim up method results in an increase in PR sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim up method results in at least a 2-fold increase in PR sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 2-fold increase in PR processed sperm compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 3-fold increase in PR processed sperm compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3.5-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 3.4-fold increase in PR processed sperm compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in sperm with a decrease in cell permeability.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in sperm with a decrease in cell permeability.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC and the swim-up method results sperm with a decrease in cell permeability.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 2-fold decrease in cell permeability.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 2.75-fold decrease in cell permeability.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in a decrease in apoptotic sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in a decrease in apoptotic sperm.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in apoptotic sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method result in at least a 2-fold decrease in apoptotic sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2-fold decrease in apoptotic processed sperm compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2.75-fold decrease in apoptotic processed sperm compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in sperm with an increase in MMP.
- ART assisted reproductive technologies
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in sperm with an increase in MMP.
- ART assisted reproductive technologies
- DGC density gradient centrifugation
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with an increase in MMP.
- separation techniques such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with an increase in MMP.
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 1.5-fold increase in MMP.
- separation techniques such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 1.5-fold increase in MMP.
- the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in processed sperm with at least a 1.5-fold increase in MMP compared to unprocessed sperm.
- ART assisted reproductive technologies
- the method is a method of diagnosing infertility.
- the method is a method of diagnosing infertility, wherein poor sperm health is indicative of infertility.
- the patient is a mammal.
- the patient is non-human.
- the patient is non-human, such as bovine, equine, canine, feline, porcine, ovine and ursidae animals.
- the patient is human.
- the patient is non-diabetic human.
- the patient is non-diabetic, non-smoking human.
- the patient is non-diabetic, non-smoking, non-obese human.
- the patient is non-diabetic, non-smoking, non-obese (with a BMI ⁇ 29.9) human.
- the patient is non-diabetic, non-smoking, non-obese (with a BMI ⁇ 29.9) man.
- the method is a method of diagnosing infertility, the method comprising: providing a biological sample from a patient; measuring the quantitative level of a biomarker in the sample; and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- mRAGE membrane-bound receptor for advanced glycation end products
- the method is a method of diagnosing infertility, the method comprising: providing a semen sample from a patient; measuring the quantitative level of a biomarker in the sample; and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- mRAGE membrane-bound receptor for advanced glycation end products
- a method of treating infertility comprising the steps of:
- the method of treating infertility comprises the steps of:
- the method of treating infertility comprises the steps of:
- FIG. 1 illustrates that RAGE is found at the acrosome and equatorial region of the human sperm cell membrane
- FIG. 2 illustrates flow cytometry gating strategy for analysing RAGE protein membrane expression and molecular markers of spermatozoa cell health
- FIG. 3 illustrates flow cytometry spermatozoa molecular health assay validation
- FIG. 4 illustrates flow cytometry and CASA evaluation of the direct swim-up preparation assay for separating PR motile sperm from washed spermatozoa
- FIG. 5 illustrates RAGE expression follows Gaussian distribution in spermatozoa from a population of non-diabetic, non-smoking and non-obese men;
- FIG. 6 illustrates RAGE is a stably expressed protein on sperm that associates with the immotile sperm population
- FIG. 7 illustrates flow cytometry quantification of sperm cell health in washed and purified spermatozoa
- FIG. 8 illustrates RAGE expression on human spermatozoa associates with spermatozoa with low mitochondrial membrane potential, dying and dead sperm with permeable cell membranes
- FIG. 9 illustrates RAGE expression correlates with the degree of DNA fragmentation in human spermatozoa.
- FIG. 10 illustrates evaluation of sperm health and RAGE protein expression in human sperm
- FIG. 11 illustrates evaluation of RAGE protein expression and sperm cell health in unprocessed and processed human sperm
- FIG. 12 illustrates evaluation of correlation between RAGE protein expression and sperm motility after processing via DGC or Swim-Up methods
- FIG. 13 illustrates evaluation of two motility assessment methods and their correlation with sperm Morphology
- FIG. 14 illustrates evaluation of mRAGE protein expression, sperm health and sperm morphology.
- the inclusion criteria were samples from normozoospermic participants undergoing a fertility assessment.
- the exclusion criteria were samples from participants with type I or type II diabetes, smokers and/or individuals with a BMI>29.9.
- Semen samples were collected after 2-5 days sexual abstinence. Samples were collected in sterile plastic containers (Starstedt, 75-562-105) and allowed to liquefy for 30 minutes at 37° C. Semen analysis was performed within 1 h of ejaculation. All samples were subject to conventional analyses for semen volume (ml), sperm concentration ( ⁇ 10 6 /ml), sperm morphology and motility, according to World Health Organisation (WHO) recommendations (WHO, 5 th Edition).
- WHO World Health Organization
- sperm morphology assessments were carried out using the simple normal/abnormal classification. Pre-stained slides (TestSimplet, Waldeck) were used following strict Kruger (Tygerberg) criteria. Briefly, a small drop of semen was added to the centre of the coverslip, placed carefully onto the pre-stained area of the slide and incubated for 15 minutes at RT. A small drop of oil was placed on the centre of the coverslip and the 100 ⁇ oil immersion lens on an Eclipse E400 microscope was used to assess morphology. Approximately 200 sperm were assessed and the percentage of normal (score >4% normal) or abnormal (score of ⁇ 4% normal) forms were determined.
- Semen samples were purified by density gradient centrifugation (DGC) or by direct swim-up.
- DGC density gradient centrifugation
- the puresperm 40/80 Nidacon PS40-100/PS80-100 separation media as used.
- a volume of 1.5 ml of 40% lower layer separation media was added to a 15 ml falcon tube followed by 1.5 ml of 80% upper layer separation media.
- a minimum volume of 1 ml of the semen sample was loaded onto the gradient and centrifuged at 400 ⁇ g for 20 minutes at RT.
- Approximately 2.5 ml of the upper layer was gently removed using a sterile Pasteur pipette and the sperm pellet was washed twice with 2 ml sperm wash media, centrifuged at 380 ⁇ g for 5 minutes at RT.
- MMP mitochondrial membrane potential
- DAPI 4,′,6-diamidino-2-phenylindole
- Annexin V-PeCy7 ThermoFisher
- Cell permeability was evaluated by incubation with 35 ng/ml DAPI (Invitrogen, D1306) for 5 minutes at 37° C. in 0.1 ml PBS with 1% FBS, protected from light. Samples were washed twice with 0.5 ml of PBS with 1% FBS prior to flow cytometry analysis. 1 ⁇ 10 6 sperm were washed into 0.5 ml of 1 ⁇ Annexin V binding buffer prior to evaluating apoptosis and necrosis using 5 ⁇ l of Annexin V-PE-Cy7 (Invitrogen; 8007-72) in 0.1 ml 1 ⁇ binding buffer and incubating for 15 minutes at RT.
- DAPI Invitrogen, D1306
- Samples were washed twice with 0.5 ml of PBS with 1% FBS prior to flow cytometry analysis. 1 ⁇ 10 6 sperm were washed into 0.5 ml of 1 ⁇ Annexin V binding buffer prior to evaluating apoptosis and necros
- RAGE protein expression was determined using a monoclonal mouse anti-human RAGE antibody-AF647 (Santa Cruz; 80652). Briefly, 1 ⁇ 10 6 spermatozoa were washed into PBS with 1% FBS prior to incubating with 2 ⁇ g/ml of RAGE antibody for 1 hour at 37° C., protected from light. Samples were washed twice with 0.5 ml of PBS with 1% FBS prior to flow cytometry analysis.
- TUNEL deoxynucleotidyl transferase dUTP nick-end labelling
- TdT TUNEL reaction TdT enzyme, BrdU, reaction buffer
- mouse anti-BrdU-AF488 antibody Invitrogen
- DAPI was measured using the 450/50 band pass filter, CMX Ros using the 610/20 filter, PE-Cy7 using the 780/60 filter, AF647 using the 670/14 filter and AF488 using the 530/30 filter.
- Spermatozoa were gated using FSC-A and SSC-A in the flame-shaped region to remove debris and cells other than spermatozoa. Doublets were omitted using the FSC-A and FSC-W dot-plot. Each fluorophore was selected and gated against FSC-A with rectangle gates used to select negative and positive populations. A quadrant plot was used to analyse apoptotic and necrotic sperm cells.
- RAGE exists in numerous forms (membrane-bound, soluble or secretory), but detection of membrane bound RAGE (mRAGE) on human spermatozoa was central to this study.
- FIG. 1 ( b ) is a Western blot analysis of RAGE expression in total, cytosolic and membrane protein lysates from transfected and non-transfected HEK293T cells using a monoclonal mouse anti-human RAGE primary antibody and a mouse anti-HRP secondary antibody.
- FIG. 1 ( c ) is a greyscale rendered confocal image showing mRAGE localisation at the acrosome and equatorial region on human sperm cells.
- FIG. 2 shows dot plots of the gating strategy for analysing mRAGE protein expression and sperm health in human sperm samples using Flow Cytometry:
- Polygonal gate drawn around sperm cell population.
- Rectangle gate drawn around single cells omitting doublets.
- Rectangle gates drawn around mRAGE positive and negative single cell population.
- Rectangle gates drawn around DAPI negative, dim and bright single cell population.
- FIG. 3 shows flow cytometry spermatozoa molecular health assay validation.
- FIG. 3 ( b ) is a bar chart showing nuclear staining in non-fixed and fixed human spermatozoa incubated with 35 ng/ml DAPI.
- FIG. 3 ( d ) is a bar chart showing mean fluorescent intensity (MFI) of MitoTracker CMX Ros positive human spermatozoa in the presence or absence of mitochondrial electron transport chain (ETC) inhibitors; FCCP, Rotenone and Malonate.
- FIG. 3 ( e ) is a representative dot plot showing human spermatozoa gating strategy for TUNEL assay (top panels) and the corresponding flow cytometry dot plots for TUNEL negative and positive control reactions (bottom panels) as indicated.
- FIG. 3 ( f ) is a bar chart showing TUNEL positive spermatozoa in negative ( ⁇ TdT enzyme) and positive (+DNasel) control reactions for analysis of DNA fragmentation in human spermatozoa
- (e) apoptosis in washed spermatozoa versus the pellet and supernatant samples obtained during a direct swim-up preparation (n 8; % mean ⁇ SEM).
- mRAGE is a Stably Expressed Protein onsperm that Associates with the Immotile Sperm Population
- FIG. 6 ( b ) is a dot plot showing mRAGE expression in DGC-purified spermatozoa over time (0-72 h).
- FIG. 6 ( b ) is a dot plot showing mRAGE expression in DGC
- FIG. 6 ( e ) is a dot plot showing mRAGE expression in washed, DGC and direct swim-up purified sperm samples.
- FIG. 7 ( b ) shows typical dot plots showing DAPI, MitoTracker CMXRos and AnnexinV fluorescence in washed spermatozoa and DGC and swim-up preparations.
- FIG. 8 ( a ) shows typical dot plots showing mRAGE positive and negative subgroups in washed spermatozoa and MitoTracker (MMP), AnnexinV-DAPI (apoptosis or necrosis) and DAPI (cell permeability) fluorescence in mRAGE positive and negative spermatozoa subgroups.
- FIG. 8 ( a ) shows typical dot plots showing mRAGE positive and negative subgroups in washed spermatozoa and MitoTracker (MMP), AnnexinV-DAPI (apoptosis or necrosis) and DAPI (cell permeability) fluorescence in
- the data generated confirms that mRAGE protein expression is higher in (i) dead and dying cells, (ii) sperm cells with low mitochondrial membrane potential, (iii) sperm cells dying by apoptosis and (iv) sperm cells dying by necrosis. It would suggest that the mRAGE membrane receptor represents a cell surface marker of an unhealthy sperm cell.
- FIG. 10 ( a ) There was a clear distinction between cell permeability, MMP and apoptosis and necrosis in mRAGE positive and negative cell populations when analysing the flow cytometry dot plots.
- FIG. 10 ( b ) Statistical analysis revealed a 3-fold increase in DAPI staining, representing non-viable cells was found in mRAGE positive cell population ( FIG. 10 ( b ) , P ⁇ 0.0001) compared to mRAGE negative cell populations.
- a 10-fold increase in cells with high MMP was found in mRAGE negative cell populations ( FIG. 10 ( b ) , P ⁇ 0.0001) compared to mRAGE positive cell populations.
- Corresponding unprocessed (neat) fractions for each processing methods included. Paired one-way ANOVA; **** P ⁇ 0.0001.
- FIG. 11 ( b ) A 5-fold and 4-fold decrease in RAGE protein expression ( FIG. 11 ( b ) ; P ⁇ 0.0001) was observed in the DGC and Swim-Up purified sperm.
- a 3.5- and 3.4-fold increase in progressively motile sperm FIG. 11 ( c ) ; P ⁇ 0.0001 in DGC and Swim-Up purified sperm.
- an average 2.75-fold decrease in impaired cell permeability, 2.75-fold decrease in apoptotic sperm and 1.5-fold increase in MMP FIG. 11 ( d ) ; P ⁇ 0.0001) was observed in the DGC and swim-up purified sperm.
- sperm purified using both methods discussed here result in highly motile progressive, live sperm that have impermeable cell membranes and highly active mitochondria.
- the decrease in mRAGE protein expression in the purified fractions would suggest that mRAGE expressing sperm are removed during the purification process.
- Mean Fluorescent Intensity (MFI; geometric mean) was analysed in sperm cells that are mRAGE positive vs mRAGE negative, and MFI was analysed in sperm cells with High MMP vs low MMP.
- Table. 1 shows the percentage of sperm cells that are mRAGE and mRAGE. Also shown is the MFI (geometric mean) of the sperm cells that are mRAGE positive and mRAGE negative. Also shown is the fold difference between the MFI of mRAGE positive vs mRAGE negative sperm cells. The average fold difference in MFI between mRAGE positive vs mRAGE negative sperm cells is 21.1% +/ ⁇ 13.8%
- Table. 1 shows the percentage of sperm cells with high MMP and low MMP. Also shown is the MFI (geometric mean) of the sperm cells that have high MMP vs low MMP. Also shown s the fold difference between the MFI of sperm cells with high MMP and low MMP. The average fold difference in MFI between cells with high vs low MMP is 25.3% +/ ⁇ 10.1%.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pregnancy & Childbirth (AREA)
- Optics & Photonics (AREA)
- Reproductive Health (AREA)
- Biophysics (AREA)
- Gynecology & Obstetrics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- General Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to a method of evaluating sperm health and identifying sperm to be used in Assisted Reproductive Technologies by providing a biological sample from a patient, preferably a semen sample; measuring the quantitative level of a biomarker in the sample; and evaluating sperm health based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
Description
- A method of evaluating sperm health and identifying sperm to be used in Assisted Reproductive Technologies
- The present invention relates to a method of evaluating sperm health and identifying sperm to be used in Assisted Reproductive Technologies by providing a biological sample from a patient, preferably a semen sample; measuring the quantitative level of a biomarker in the sample; and evaluating sperm health based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- Infertility affects approximately 15% of couples that are of reproductive age and approximately 30-40% of cases can be attributed to male factor infertility. Male infertility can arise from a variety of situations including varicocele, genetic alterations, systemic diseases, and altered semen parameters. However, the single greatest contributory factor to male infertility is sperm damage due to oxidative stress and resultant DNA fragmentation, estimated to account for between 30% and 80% of cases. Not only does DNA fragmentation affect sperm quality, it has also been found to negatively impact embryo quality, implantation rates and miscarriage rates.
- Current investigations of male infertility include an evaluation of the physical characteristics of a semen sample (volume, viscosity, pH and colour) and a microscopic evaluation of sperm in a sample (concentration, motility, morphology and vitality). These tests provide a functional and physical read out of the health of the sperm population in a semen sample. Assessment of double-stranded breaks in sperm DNA is recommended to provide additional information about the molecular health of sperm, fertilization rates and DNA integrity. Tests include the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-nick-end labelling (or TUNEL), comet, or sperm chromatin dispersion (SCD) assays. Unfortunately, these tests are often not included in investigations of male infertility as they are expensive, time-consuming, invasive and do not adequately report on sperm health.
- Extracellular markers that correlate with the degree of sperm DNA damage and the quality of the sperm would be highly valuable for the identification and removal of damaged sperm prior to assisted reproductive techniques (ART). Significant efforts have been made over the last three decades to identify a more sophisticated marker. Mainly intracellular markers of DNA fragmentation, oxidative DNA damage, oxidative stress, mitochondrial function and sperm cell death have been identified. However, these require invasive internal labelling procedures leaving the sperm non-viable afterwards. In this study, we evaluate an extracellular marker of sperm cell health.
- The Receptor for Advanced Glycation End products (RAGE) is a central signalling molecule of the innate immune system and belongs to a class of cell-surface, pattern recognition receptors encoded at the MHC class III locus. It exists in numerous forms as membrane-bound (full length), soluble or secretory protein. Soluble RAGE (sRAGE) is produced by both proteolytic cleavage of membrane-bound/full length RAGE (mRAGE/fl-RAGE), and alternative mRNA splicing. The soluble isoforms include the extracellular domains but lack the transmembrane and cytoplasmic domains
- RAGE is long established as a key contributor to severe chronic pathologies including neurodegeneration, atherosclerosis, cardiovascular diseases, osteoarthritis and diabetes. Under normal conditions, the expression of RAGE is very low. However, during disease, the production of RAGE is highly induced on the cell surface by a number of different ligands such as advanced glycation end products (AGE), high mobility group box-1 (HMGB1), S100/calgranulins, phosphatidylserine, C3. In addition, RAGE expression is further enhanced after immune activation or infection and many RAGE ligands are secreted by monocytes, macrophages, neutrophils and leukocytes which are likely to be crucial for the initiation and propagation of a RAGE-dependent inflammatory response. The location of the membrane-bound receptor at the acrosomal cap of human sperm, which is a direct access point to nuclear DNA within the sperm head, has been implicated in playing a role in mediating and exacerbating nuclear DNA damage.
- This study describes mRAGE as a diagnostic biomarker of low-quality sperm. mRAGE expression was analysed on sperm from non-diabetic, non-obese and non-smoking men thereby removing the heightened effects of RAGE and its ligands on sperm health in these conditions. Flow cytometry was used to quantitatively assess mRAGE expression at a cellular level on sperm and computer-assisted sperm analysis (CASA) was used to evaluate sperm motility. By analysing mRAGE as well as mitochondrial membrane potential (MMP), cell permeability, motility, morphology, apoptosis and DNA fragmentation at the cellular level, strongest evidence is provided for a direct relationship.
- It was observed that mRAGE expression on sperm correlates with mitochondrial dysfunction, cell death, DNA fragmentation and immotile sperm. In addition, purifying sperm using conventional techniques (density gradient centrifugation and direct swim-up) allowed for the removal of mRAGE expressing spermatozoa. Thus, there remains a need for a reliable, less invasive, extracellular marker of evaluating sperm health. As RAGE is membrane bound, it is also possible to use it as a means to remove the damaged sperm.
- According to a first aspect of the present invention, there is provided a method of evaluating sperm health; the method comprising the steps of:
-
- (a) providing a biological sample from a patient;
- (b) measuring the quantitative or qualitative level of a biomarker in the sample; and
- (c) evaluating sperm health based on the quantitative or qualitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- Optionally, the method of evaluating sperm health is an in vitro method.
- Optionally, mRAGE is bound to a membrane of a cell.
- Optionally, mRAGE is bound to the membrane of a sperm.
- Optionally, mRAGE is bound to the membrane of the sperm head.
- Optionally, mRAGE is bound to the membrane of the equatorial regions of the sperm head.
- Optionally, mRAGE is a transmembrane receptor.
- Optionally, mRAGE comprises three extracellular domains.
- Optionally, mRAGE comprises three extracellular domains—a V-type Ig domain and two C-type Ig domains (C1 and C2).
- Optionally, mRAGE comprises three extracellular domains—a V-type Ig domain and two C-type Ig domains (C1 and C2); and a transmembrane domain.
- Optionally, mRAGE comprises three extracellular domains, a V-type Ig domain and two C-type Ig domains (C1 and C2); a transmembrane domain; and a cytoplasmic tail.
- Optionally, mRAGE is full-length RAGE (fl-RAGE).
- Preferably, the biological sample is a semen sample.
- Optionally, the biomarker is a gene. Optionally, the biomarker is a nucleic acid. Optionally, the biomarker is a deoxyribonucleic acid. Optionally, the biomarker is a ribonucleic acid. Optionally, the biomarker is a protein. Optionally, the biomarker is a peptide.
- Optionally, the biomarker for evaluating sperm health is a gene having a GenBank Accession Version Number AQY76652
- Preferably, the biomarker for evaluating sperm health is a protein having a UniProt ID Q15109
- Preferably, the measuring step (b) comprises measuring the quantitative level of mRAGE on sperm cells from a semen sample.
- Optionally, the measuring step (b) comprises measuring the quantitative level of mRAGE on sperm cells from a semen sample by any quantitative analytical technique.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample by a cell-based assay.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample by flow cytometry.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a RAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a sRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a mouse anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a rabbit anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a rat anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a sheep anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal rabbit anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal rat anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal sheep anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using a monoclonal mouse anti-human mRAGE antibody.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using the monoclonal mouse anti-human mRAGE antibody—AF647.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using DNA aptamers.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using RNA aptamers.
- Optionally, the measuring step (b) comprises measuring the quantitative or qualitative level of mRAGE on sperm cells from a semen sample and is determined using peptide aptamers.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the presence of mRAGE is defined as mRAGE positive.
- Optionally, the evaluating step (c) comprises evaluating the presence or absence of quantifiable levels of mRAGE on sperm cells from a semen sample to define the patient as mRAGE negative or mRAGE positive.
- Optionally, the presence of quantifiable levels of mRAGE on sperm cells from the semen sample defines the patient as mRAGE positive and wherein the absence of quantifiable levels of mRAGE on sperm cells from the semen sample defines the patient as mRAGE negative.
- Optionally, the evaluating step (c) comprises comparing the amount of sperm expressing quantifiable levels of mRAGE to the amount of sperm not expressing quantifiable levels of mRAGE.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 1-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 2-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 3-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 4-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 5-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 6-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 7-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 8-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 9-fold.
- Optionally, the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 10-fold.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample to define the patient as mRAGE negative or mRAGE positive.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE negative is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is lower than 10-fold compared to the mRAGE positive sperm cells.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing qualifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 10-fold higher compared to the mRAGE negative sperm cells.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 20-fold higher compared to the mRAGE negative sperm cells.
- Optionally, the evaluating step (c) comprises evaluating the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is at least 30-fold higher compared to the mRAGE negative sperm cells.
- Optionally, a Mean Fluorescent Intensity value expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value not expressing quantifiable levels of mRAGE by less than 10-fold.
- Optionally, a Mean Fluorescent Intensity value expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value not expressing quantifiable levels of mRAGE by at least 10-fold.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is indicative of poor sperm health.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE negative is indicative of healthy, fertile sperm.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of abnormal sperm.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of normal sperm.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of good sperm health.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE negative is indicative of one or more of normal sperm and good sperm health.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of one or more of poor sperm health and abnormal sperm.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of poor sperm motility.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm and poor sperm motility.
- Further optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of infertility.
- Optionally, the evaluating step (c) comprises evaluating sperm health based on the quantitative or qualitative level of mRAGE on sperm cells from a semen sample, wherein the mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm, poor sperm motility and infertility.
- Optionally, in the method of evaluating sperm health, the patient is a mammal.
- Optionally, in the method of evaluating sperm health, the patient is non-human
- Optionally, in the method of evaluating sperm health, the patient is non-human, such as bovine, equine, canine, feline, porcine, ovine and ursidae animals.
- Preferably, in the method of evaluating sperm health, the patient is human.
- Optionally, in the method of evaluating sperm health, the patient is non-diabetic human.
- Optionally, in the method of evaluating sperm health, the patient is non-diabetic, non-smoking human.
- Optionally, in the method of evaluating sperm health, the patient is non-diabetic, non-smoking, non-obese human.
- Optionally, in the method of evaluating sperm health, the patient is non-diabetic, non-smoking, non-obese (BMI<29.9) human.
- Optionally, in the method of evaluating sperm health, the patient is non-diabetic, non-smoking, non-obese (BMI<29.9) man.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein the sperm are categorised as immotile (IM), non-progressively motile (NP) or progressively motile (PR).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health and poor sperm motility.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as IM do not move, which is indicative of poor sperm health, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as NP move, but do not move in a substantially linear direction.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as NP move, but do not move in a substantially linear direction, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as PR move in a substantially linear direction.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein sperm categorised as PR move in a substantially linear direction, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 10% of sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 20% of sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 30% of sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 32% of sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 32% of sperm categorised as PR in a semen sample is considered normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 10% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 20% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 30% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 40% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein at least 40% of a combination of sperm categorised as NP and sperm categorised as PR in a semen sample is considered normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm motility, wherein the percentage of mRAGE positive sperm is inversely correlated to the percentage of PR sperm; defined as sperm with a space gain of ≥5 μm s−1.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein the sperm are classified as normal or abnormal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein the sperm are classified as normal or abnormal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; and there is a well-defined acrosomal region comprising 40-70% of the sperm head area.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; and there is a well-defined acrosomal region comprising 40-70% of the sperm head area, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; and the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; and the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; and the midpiece of the sperm is slender, regular and substantially the same length as the sperm head.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; and the midpiece of the sperm is slender, regular and substantially the same length as the sperm head, as by the defined World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; and the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; and the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head; and the principal piece has a uniform calibre along its length, is thinner than the midpiece, and is approximately 45 micrometres long.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; there is a well-defined acrosomal region comprising 40-70% of the sperm head area; the acrosomal region contains no large vacuoles, and not more than two small vacuoles, which does not occupy more than 20% of the sperm head; the midpiece of the sperm is slender, regular and substantially the same length as the sperm head; the major axis of the midpiece of the sperm is aligned with the major axis of the sperm head; and the principal piece has a uniform calibre along its length, is thinner than the midpiece, and is approximately 45 micrometres long, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing sperm morphology, wherein sperm are classified as abnormal when there is deviation from normal, which is indicative of poor sperm health, as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises a step of assessing mitochondrial membrane potential (MMP).
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros does not stain cells with low MMP; as defined as a Mean Fluorescent Intensity (MFI; geometric mean) value that is 10-fold, or lower, than the mRAGE negative sperm cells, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros stains cells with high MMP.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros, wherein Mitotracker CMX Ros stains cells with high MMP, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with low MMP is present in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with low MMP is present in mRAGE positive sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and an increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to RAGE positive sperm, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold increase in sperm with high MMP is present in mRAGE negative sperm compared to mRAGE positive sperm, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 2-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 5-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to RAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises a step of assessing MMP, wherein MMP is assessed by staining with Mitotracker CMX Ros and at least a 10-fold decrease in sperm with high MMP is present in mRAGE positive sperm compared to mRAGE negative sperm.
- Optionally, quantifiable levels of MMP is indicative of mRAGE negative sperm and the absence of quantifiable levels of MMP is indicative of mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises the step of assessing cell permeability.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, and positive staining by DAPI represents non-viable cells.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein an increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein at least a 2-fold increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing cell permeability, wherein cell permeability is assessed staining with DAPI, and positive staining by DAPI represents non-viable cells, wherein at least a 3-fold increase in non-viable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, at least a 2-fold decrease in non-permeable cells is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health
- Optionally, the method of evaluating sperm health further comprises the step of assessing apoptosis.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V and positive staining with Annexin V is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and positive staining with Annexin V in combination with DAPI is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and an increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 2-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is evaluated by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 4-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V in combination with DAPI to allow for the simultaneous assessment of apoptosis and necrosis, and at least a 4-fold increase in apoptosis and necrosis is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises the step of assessing DNA fragmentation.
- Optionally, the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay.
- Optionally, the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein an increase in DNA fragmentation is present in mRAGE positive sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein an increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein at least a 2-fold increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises assessing DNA fragmentation using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, wherein at least a 3-fold increase in DNA fragmentation is present in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
- Optionally, the method of evaluating sperm health further comprises the step of identifying sperm that can be used in assisted reproductive technologies (ART).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including intrauterine insemination (IUI).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI) and intracytoplasmic injection (ICSI).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI), intracytoplasmic injection (ICSI) and in vitro fertilisation (IVF).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), including any one or more of intrauterine insemination (IUI), intracytoplasmic injection (ICSI), in vitro fertilisation (IVF) and artificial insemination (AI).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including density gradient centrifugation (DGC).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including the sperm swim-up method.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including the sperm swim-up method, as defined World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010).
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm; and the swim-up method relies on sperm motility.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method separates sperm based on their densities as morphological normal sperm have a higher density than abnormal sperm; and the swim-up method relies on sperm motility to separate PR sperm from IM sperm and NP sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in a decrease in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in a decrease in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in mRAGE positive sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in mRAGE positive sperm, which is indicative of good sperm health.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm and the swim-up method results in at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 2-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 3-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 4-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm and the swim-up method results in at least a 5-fold decrease in mRAGE positive processed sperm compared to mRAGE negative processed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DCG method results in an increase in PR sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim up method results in an increase in PR sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim up method results in an increase in PR sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim up method results in at least a 2-fold increase in PR sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 2-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 2-fold increase in PR processed sperm compared to unprocessed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 3-fold increase in PR processed sperm compared to unprocessed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in at least a 3.5-fold increase in PR processed sperm compared to unprocessed sperm and the swim-up method results in at least a 3.4-fold increase in PR processed sperm compared to unprocessed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in sperm with a decrease in cell permeability.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in sperm with a decrease in cell permeability.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC and the swim-up method results sperm with a decrease in cell permeability.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 2-fold decrease in cell permeability.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 2.75-fold decrease in cell permeability.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in a decrease in apoptotic sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in a decrease in apoptotic sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in a decrease in apoptotic sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method result in at least a 2-fold decrease in apoptotic sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2-fold decrease in apoptotic processed sperm compared to unprocessed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in at least a 2.75-fold decrease in apoptotic processed sperm compared to unprocessed sperm.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method results in sperm with an increase in MMP.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the swim-up method results in sperm with an increase in MMP.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with an increase in MMP.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in sperm with at least a 1.5-fold increase in MMP.
- Optionally, the method of evaluating sperm health further comprises identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, such as density gradient centrifugation (DGC) and the sperm swim-up method, wherein the DGC method and the swim-up method results in processed sperm with at least a 1.5-fold increase in MMP compared to unprocessed sperm.
- Optionally, the method is a method of diagnosing infertility.
- Optionally, the method is a method of diagnosing infertility, wherein poor sperm health is indicative of infertility.
- Optionally, in the method of diagnosing infertility, the patient is a mammal.
- Optionally, in the method of diagnosing infertility, the patient is non-human.
- Optionally, in the method of diagnosing infertility, the patient is non-human, such as bovine, equine, canine, feline, porcine, ovine and ursidae animals.
- Preferably, in the method of diagnosing infertility, the patient is human.
- Optionally, in the method of diagnosing infertility, the patient is non-diabetic human.
- Optionally, in the method of diagnosing infertility, the patient is non-diabetic, non-smoking human.
- Optionally, in the method of diagnosing infertility, the patient is non-diabetic, non-smoking, non-obese human.
- Optionally, in the method of diagnosing infertility, the patient is non-diabetic, non-smoking, non-obese (with a BMI<29.9) human.
- Optionally, in the method of diagnosing infertility, the patient is non-diabetic, non-smoking, non-obese (with a BMI<29.9) man.
- Optionally, the method is a method of diagnosing infertility, the method comprising: providing a biological sample from a patient; measuring the quantitative level of a biomarker in the sample; and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- Preferably, the method is a method of diagnosing infertility, the method comprising: providing a semen sample from a patient; measuring the quantitative level of a biomarker in the sample; and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
- According to a further aspect of the present invention, there is provided a method of treating infertility, the method comprising the steps of:
-
- (a) providing a biological sample from a patient;
- (b) measuring the presence or absence of quantitative level of membrane-bound receptor for advanced glycation end products (mRAGE) in the sample to define the patient as mRAGE negative or mRAGE positive; and
- (c) treating infertility by selecting the mRAGE negative samples and fertilising an egg.
- Optionally, the method of treating infertility comprises the steps of:
-
- (a) identifying sperm that can be used in assisted reproductive technologies (ART); and
- (b) treating infertility by selecting the sperm that can be used in ART and fertilising an egg.
- Optionally, the method of treating infertility comprises the steps of:
-
- (a) identifying sperm that can be used in assisted reproductive technologies (ART) by providing a semen sample from a patient;
- (b) measuring the presence or absence of quantitative level of membrane-bound receptor for advanced glycation end products (mRAGE) in the semen sample to define the patient as mRAGE negative or mRAGE positive; and
- (c) treating infertility by selecting the mRAGE negative sperm and fertilising an egg with the selected sperm.
- Embodiments of the present invention will now be described with reference to the accompanying drawings and following non-limiting examples, in which:
-
FIG. 1 illustrates that RAGE is found at the acrosome and equatorial region of the human sperm cell membrane; -
FIG. 2 illustrates flow cytometry gating strategy for analysing RAGE protein membrane expression and molecular markers of spermatozoa cell health; -
FIG. 3 illustrates flow cytometry spermatozoa molecular health assay validation; -
FIG. 4 illustrates flow cytometry and CASA evaluation of the direct swim-up preparation assay for separating PR motile sperm from washed spermatozoa; -
FIG. 5 illustrates RAGE expression follows Gaussian distribution in spermatozoa from a population of non-diabetic, non-smoking and non-obese men; -
FIG. 6 illustrates RAGE is a stably expressed protein on sperm that associates with the immotile sperm population; -
FIG. 7 illustrates flow cytometry quantification of sperm cell health in washed and purified spermatozoa; -
FIG. 8 illustrates RAGE expression on human spermatozoa associates with spermatozoa with low mitochondrial membrane potential, dying and dead sperm with permeable cell membranes; -
FIG. 9 illustrates RAGE expression correlates with the degree of DNA fragmentation in human spermatozoa. -
FIG. 10 illustrates evaluation of sperm health and RAGE protein expression in human sperm; -
FIG. 11 illustrates evaluation of RAGE protein expression and sperm cell health in unprocessed and processed human sperm; -
FIG. 12 illustrates evaluation of correlation between RAGE protein expression and sperm motility after processing via DGC or Swim-Up methods; -
FIG. 13 illustrates evaluation of two motility assessment methods and their correlation with sperm Morphology; and -
FIG. 14 illustrates evaluation of mRAGE protein expression, sperm health and sperm morphology. - This study was carried out using semen samples from anonymous patients attending Merrion Fertility Clinic with permission from the donors and ethical approval from the School of Medicine Research Ethics Committee, Trinity College Dublin and the National Maternity Hospital, Holles Street, Ethics Committee. The research was carried out at Trinity Biomedical Sciences Institute, Trinity College Dublin.
- Men attending the Merrion Fertility Clinic between August 2017 and July 2018 were invited to participate in this project and written consent was received from all participants. The inclusion criteria were samples from normozoospermic participants undergoing a fertility assessment. The exclusion criteria were samples from participants with type I or type II diabetes, smokers and/or individuals with a BMI>29.9.
- Semen samples were collected after 2-5 days sexual abstinence. Samples were collected in sterile plastic containers (Starstedt, 75-562-105) and allowed to liquefy for 30 minutes at 37° C. Semen analysis was performed within 1 h of ejaculation. All samples were subject to conventional analyses for semen volume (ml), sperm concentration (×106/ml), sperm morphology and motility, according to World Health Organisation (WHO) recommendations (WHO, 5th Edition).
- Sperm morphology assessments were carried out using the simple normal/abnormal classification. Pre-stained slides (TestSimplet, Waldeck) were used following strict Kruger (Tygerberg) criteria. Briefly, a small drop of semen was added to the centre of the coverslip, placed carefully onto the pre-stained area of the slide and incubated for 15 minutes at RT. A small drop of oil was placed on the centre of the coverslip and the 100× oil immersion lens on an Eclipse E400 microscope was used to assess morphology. Approximately 200 sperm were assessed and the percentage of normal (score >4% normal) or abnormal (score of <4% normal) forms were determined.
- Sperm motility assessments were carried out by CASA using the Hamilton Thorne software and a Zeiss microscope, following WHO and manufacturers' guidelines. Briefly, semen samples were resuspended using Pasteur pipettes and diluted 1 in 3 in sperm wash media. A volume of 3 μl of diluted semen was added to the CASA microscope slide, placed onto the pre-heated stage (37° C.) and allowed to settle until no drift was apparent on the screen. Motility assessment was evaluated by capturing at least 1000 sperm cell tracks per slide for unprocessed samples and 300 for processed samples. Total cell count (×106 /ml), percentage immotile (IM), non-progressively motile (NP) and progressively motile (PR) sperm and average path velocity (VAP) for sperm was recorded for each sample.
- Seminal plasma was removed by washing spermatozoa into 0.5 ml of sperm wash media (Irvine Scientific, 9983) by centrifugation at 1,500×g for 1 minute at RT, repeated twice. The final sperm pellet was resuspended in 0.1 ml sperm wash media prior to downstream experiments. The sample is hereafter referred to as “washed spermatozoa”.
- Semen samples were purified by density gradient centrifugation (DGC) or by direct swim-up. For DGC, the
puresperm 40/80 Nidacon PS40-100/PS80-100 separation media as used. A volume of 1.5 ml of 40% lower layer separation media was added to a 15 ml falcon tube followed by 1.5 ml of 80% upper layer separation media. A minimum volume of 1 ml of the semen sample was loaded onto the gradient and centrifuged at 400×g for 20 minutes at RT. Approximately 2.5 ml of the upper layer was gently removed using a sterile Pasteur pipette and the sperm pellet was washed twice with 2 ml sperm wash media, centrifuged at 380×g for 5 minutes at RT. The washed pellet, containing highly motile purified sperm, was resuspended in 0.5 ml of sperm wash media. For direct swim-up, a minimum of 1 ml of seminal plasma was washed twice in 2 ml sperm wash media by centrifugation at 380×g for 5 minutes at RT. The washed sperm pellet was gently resuspended and layered underneath a 1 ml volume of multipurpose handling media (Irvine Scientific, 90166, pre-equilibrated at 37° C. and incubated for 30 minutes at RT. Approximately 0.8 ml of the top layer was removed containing highly motile purified sperm. - Measurement of mitochondrial membrane potential (MMP), cell permeability and cell death were determined using MitoTracker CMX Ros (ThermoFisher), DAPI (4′,6-diamidino-2-phenylindole; ThermoFisher) and Annexin V-PeCy7 (ThermoFisher), respectively. Briefly, 1×106 sperm from seminal plasma were washed into 0.5 ml of sperm wash media followed by a two-step wash into 0.5 ml PBS with 1% FBS. MMP was evaluated by incubation with 6.25 nM Mitotracker CMX Ros (Invitrogen; M7512) for 15 minutes at 37° C. Cell permeability was evaluated by incubation with 35 ng/ml DAPI (Invitrogen, D1306) for 5 minutes at 37° C. in 0.1 ml PBS with 1% FBS, protected from light. Samples were washed twice with 0.5 ml of PBS with 1% FBS prior to flow cytometry analysis. 1×106 sperm were washed into 0.5 ml of 1× Annexin V binding buffer prior to evaluating apoptosis and necrosis using 5 μl of Annexin V-PE-Cy7 (Invitrogen; 8007-72) in 0.1 ml 1× binding buffer and incubating for 15 minutes at RT.
- Measurement of RAGE protein expression was determined using a monoclonal mouse anti-human RAGE antibody-AF647 (Santa Cruz; 80652). Briefly, 1×106 spermatozoa were washed into PBS with 1% FBS prior to incubating with 2 μg/ml of RAGE antibody for 1 hour at 37° C., protected from light. Samples were washed twice with 0.5 ml of PBS with 1% FBS prior to flow cytometry analysis.
- Measurement of DNA fragmentation was determined using the ApoBrdU-terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, following manufacturer's guidelines (Invitrogen; A23210) with minor modifications. Briefly, after washing in PBS with 1% FBS, 5×106 washed sperm cells were fixed with 4% PFA for 30 minutes and washed twice using PBS with 1% FBS. Fixed cells were permeabilised by incubating in 0.1 ml 0.1% Triton-X, 0.1% sodium citrate in PBS for 2 minutes on ice and washed twice with 0.5 ml of wash buffer, supplied with the kit. Fixed and permeabilised cells were incubated with a TdT TUNEL reaction (TdT enzyme, BrdU, reaction buffer) for 1 hour at 37° C., washed twice with 0.5 ml of rinse buffer and incubated with the mouse anti-BrdU-AF488 antibody (Invitrogen) for 30 mins at RT. Cells were washed twice with 0.5 ml PBS with 1% FBS and counterstained with 10 μl of PI in RNaseA buffer, supplied with the kit.
- For flow cytometry data collection, a minimum of 10,000 single events were recorded for each sample at a flow rate of 200 events/s using the BD LSR Fortessa flow cytometer. DAPI was measured using the 450/50 band pass filter, CMX Ros using the 610/20 filter, PE-Cy7 using the 780/60 filter, AF647 using the 670/14 filter and AF488 using the 530/30 filter. Spermatozoa were gated using FSC-A and SSC-A in the flame-shaped region to remove debris and cells other than spermatozoa. Doublets were omitted using the FSC-A and FSC-W dot-plot. Each fluorophore was selected and gated against FSC-A with rectangle gates used to select negative and positive populations. A quadrant plot was used to analyse apoptotic and necrotic sperm cells.
- For microscopic evaluation of RAGE and mitochondrial localisation on sperm, slides were viewed using a Leica SP8 Gated STED confocal microscope. For RAGE localisation analysis, 1×106 washed unprocessed human sperm cells were incubated with 4 μg/ml mouse anti-human RAGE primary antibody (Santa Cruz; sc-80652) for 1 hour at 37° C. followed by 2 μg/ml goat anti-mouse AF647 secondary antibody (ThermoFisher; A28181) for 30 minutes at 37° C. For mitochondrial localisation, 1×106 purified human sperm cells (via DGC) were incubated with 0.5 μM MitoTracker CMX Ros for 15 minutes at 37° C. Stained cells were fixed with 0.5
ml 4% PFA pH 7.2 for 20 minutes at RT and incubated with 70 ng/ml DAPI. Slides were prepared by adding 20 μl of stained sperm cells onto poly-L-lysine microscope slides and air-dried at 37° C. prior to adding mounting medium and a coverslip. - Statistical analyses were performed using Prism 6.0 software (GraphPad). Morphology and mRAGE protein expression were compared using parametric unpaired t-test. Motility and RAGE protein expression were compared using parametric paired t-test. Flow cytometry markers were compared using parametric paired t-test. All data was assumed to be normally distributed. Values are expressed as ±SD. A P value of <0.05 is considered as statistically significant. The Pearson's correlation test was performed to analyse the correlation between RAGE protein expression and motility. A one-way ANOVA test, with a 2-tailed P value, was used to compare flow cytometry marker expression across unprocessed and processed samples. A P value of <0.05 is considered as statistically significant.
- RAGE exists in numerous forms (membrane-bound, soluble or secretory), but detection of membrane bound RAGE (mRAGE) on human spermatozoa was central to this study.
-
FIG. 1(a) is a bar chart showing mRAGE positive fluorescence in non-transfected and transfected HEK293T cells analysed with monoclonal RAGE antibody using flow cytometry (n=2 biological replicates, SD).FIG. 1(b) is a Western blot analysis of RAGE expression in total, cytosolic and membrane protein lysates from transfected and non-transfected HEK293T cells using a monoclonal mouse anti-human RAGE primary antibody and a mouse anti-HRP secondary antibody.FIG. 1(c) is a greyscale rendered confocal image showing mRAGE localisation at the acrosome and equatorial region on human sperm cells. - In summary, using a transiently transfected HEK293T cell model, a number of antibodies were screened for mRAGE detection using flow cytometry (
FIG. 1(a) ) and mRAGE detection was demonstrated using Western blot (FIG. 1(b) ). The mouse anti-human mRAGE antibody was also capable of detecting mRAGE at the equatorial mid-piece and acrosomal region of the sperm cell head (FIG. 1(c) ). -
FIG. 2 shows dot plots of the gating strategy for analysing mRAGE protein expression and sperm health in human sperm samples using Flow Cytometry: (i) Polygonal gate drawn around sperm cell population. (ii) Rectangle gate drawn around single cells omitting doublets. (iii) Rectangle gates drawn around mRAGE positive and negative single cell population. (iv) Rectangle gates drawn around DAPI negative, dim and bright single cell population. (v) Rectangle gates drawn around MitoTracker positive (high MMP) and negative (low MMP) single cell population. (vi) Quadrant plots analyzing apoptosis and necrosis: Q1:AnV-DAPI+ (Necrotic); Q2:AnV-FDAPI+ (Apoptotic); Q3:AnV+DAPI-(Early apoptotic); Q4:AnV-DAPI− (live). -
FIG. 3 shows flow cytometry spermatozoa molecular health assay validation.FIG. 3(a) is a line graph showing human spermatozoa incubated with increasing concentrations of DAPI (n=3). Final concentration selected for further studies is indicated by the boxed region.FIG. 3(b) is a bar chart showing nuclear staining in non-fixed and fixed human spermatozoa incubated with 35 ng/ml DAPI.FIG. 3(c) is a line graph showing human spermatozoa incubated with increasing concentrations of MitoTracker CMX Ros (n=3). Final concentration selected for further studies is indicated by the boxed region.FIG. 3(d) is a bar chart showing mean fluorescent intensity (MFI) of MitoTracker CMX Ros positive human spermatozoa in the presence or absence of mitochondrial electron transport chain (ETC) inhibitors; FCCP, Rotenone and Malonate.FIG. 3(e) is a representative dot plot showing human spermatozoa gating strategy for TUNEL assay (top panels) and the corresponding flow cytometry dot plots for TUNEL negative and positive control reactions (bottom panels) as indicated.FIG. 3(f) is a bar chart showing TUNEL positive spermatozoa in negative (−TdT enzyme) and positive (+DNasel) control reactions for analysis of DNA fragmentation in human spermatozoa - Flow cytometry and CASA evaluation of the direct swim-up preparation assay for separating PR motile sperm from washed spermatozoa. Comparison of percentages of:
FIG. 4(a) mRAGE protein expression,FIG. 4(b) PR motility,FIG. 4(c) cell permeability,FIG. 4(d) MMP andFIG. 4 . (e) apoptosis in washed spermatozoa versus the pellet and supernatant samples obtained during a direct swim-up preparation (n=8; % mean±SEM). One-way ANOVA **** P<0.0001, **=P<0.005; *=P<0.05. -
FIG. 5 shows a histogram of frequency distribution of mRAGE expression in n=60 washed sperm samples measured by flow cytometry. - Overall, it was found that mRAGE expression on human spermatozoa is normally distributed in the population of patients attending fertility clinics in the absence of medical or ‘self-inflicted’ conditions such as diabetes, smoking or obesity.
-
FIG. 6(a) is a line-graph showing percentage immotile sperm and mRAGE expression in DGC-purified spermatozoa measured over time (0-72 h) analysed using CASA and FC (n=6% mean±SEM).FIG. 6(b) is a dot plot showing mRAGE expression in DGC-purified spermatozoa over time (0-72 h).FIG. 6(c) is a comparison of the percentages of PR motility in washed and purified sperm samples (n=15; % mean±SEM). One-way ANOVA; ****=P<0.0001.FIG. 6(d) is a comparison of the percentages of mRAGE protein expression in washed and purified sperm samples (n=15; % mean±SEM). One-way ANOVA; ****=P<0.0001.FIG. 6(e) is a dot plot showing mRAGE expression in washed, DGC and direct swim-up purified sperm samples.FIG. 6(f) is evaluation of the correlation between % total IM spermatozoa and the percentage of mRAGE protein expression in washed spermatozoa (n=35). Pearson's correlation R2 value indicated on graph; error bars within 95% CI shown by dashed line. - Using a time-course assay, it was observed that mRAGE expression does not differ greatly (4.6%±1.3 (SD) to 14.7%±7.3) over time whereas the percentage of IM sperm in purified samples increases from 7.7±1.9% (SD) to 91.3±5.1% (SD) over 72 hours (
FIG. 6(a) and (b) , P<0.0001). This would suggest that mRAGE expression on sperm is directly related to the health status of the individual and does not change following collection of the semen sample. Specifically, the percentage of PR sperm increased from an average of 20.5±10.9% (SD) in washed samples to 69.1±11.9% (FIG. 6(c) , P<0.0001) in purified samples, for both techniques. In contrast, the percentage of mRAGE expressing spermatozoa decreased from an average of 22.1±10.1% (SD) in washed samples to 5.5±4.8% (SD) in purified samples (FIG. 6(d) and (e) , P<0.0001). In a larger cohort of samples, it was also observed that mRAGE expression correlates with poor sperm motility (FIG. 6(f) , P<0.0001) which suggests that mRAGE expression could be used to distinguish between normal and abnormal sperm at a cellular level. This data shows that the absence of mRAGE in the purified samples correlates with PR sperm and improved cell health. -
FIG. 7(a) shows comparison of the percentages of cell permeability, apoptotic cells and MMP in washed and DGC and swim-up preparations (n=15; % mean±SEM). One-way ANOVA; ****=P<0.0001.FIG. 7(b) shows typical dot plots showing DAPI, MitoTracker CMXRos and AnnexinV fluorescence in washed spermatozoa and DGC and swim-up preparations. - Concurrent analysis of the molecular parameters of spermatozoa cell health using flow cytometry revealed that mitochondrial function, the levels of cell death (via apoptosis and necrosis) and membrane integrity of spermatozoa greatly improved in the purified samples compared to washed samples (
FIG. 7 , P<0.0001). -
FIG. 8(a) shows typical dot plots showing mRAGE positive and negative subgroups in washed spermatozoa and MitoTracker (MMP), AnnexinV-DAPI (apoptosis or necrosis) and DAPI (cell permeability) fluorescence in mRAGE positive and negative spermatozoa subgroups.FIG. 8(b) shows comparison of the percentages of MitoTracker positive, AnnexinV-DAPI positive or negative and DAPI positive spermatozoa in mRAGE positive and negative spermatozoa subgroups (n=35; % mean±SEM). Paired t-test; ****=P<0.0001.FIG. 8(c) shows evaluation of the correlation between markers of sperm cell health and mRAGE protein expression in washed samples (n=35). Pearson's correlation R2 value indicated on graph; error bars within 95% CI shown by dashed line. ****=P<0.0001; **=P<0.005; *=P<0.05. - DNA integrity, mitochondrial function and levels of cell death have all been used to analyse molecular health of sperm. Data above showed an improvement to cell health and reduction in mRAGE expressing sperm in purified samples. In a larger cohort of samples, using flow cytometry, it was observed that mRAGE expression on spermatozoa is associated with unhealthy sperm shown by decreased mitochondrial function (10-fold), elevated levels of apoptosis (6-fold) and necrosis (2.5-fold) and decreased membrane integrity (3-fold) in mRAGE positive populations compared to mRAGE negative populations (
FIG. 8 , P<0.0001). This data supports the use of mRAGE as a diagnostic biomarker of unhealthy sperm. - To summarise, the data generated confirms that mRAGE protein expression is higher in (i) dead and dying cells, (ii) sperm cells with low mitochondrial membrane potential, (iii) sperm cells dying by apoptosis and (iv) sperm cells dying by necrosis. It would suggest that the mRAGE membrane receptor represents a cell surface marker of an unhealthy sperm cell.
-
FIG. 9(a) shows comparison of the percentages of TUNEL positive spermatozoa in mRAGE positive and negative subgroups (n=8; % mean±SEM). Paired t-test; ***=P<0.005.FIG. 9(b) shows evaluation of the correlation between the percentages of TUNEL positive and mRAGE positive spermatozoa (n=8). Pearson's correlation R2 value indicated; error bars within 95% CI shown by dashed line. - It was observed that the levels of DNA fragmentation, analysed using the TUNEL assay, were significantly higher (2.5-fold) in mRAGE positive spermatozoa compared tom RAGE negative spermatozoa (
FIG. 9(a) , P<0.001) in non-diabetic men. In addition, the data shows that mRAGE expression on sperm correlates with sperm DNA damage (FIG. 9(b) , P<0.05) and could be used as an indirect marker of DNA integrity and sperm cell health. - Evaluation of sperm cell health in mRAGE positive and mRAGE negative cell populations in unprocessed sperm. mRAGE protein expression was evaluated using the Mouse Anti-Human mRAGE AF647 antibody; cell permeability was evaluated using DAPI; MMP was evaluated using MitoTracker CMX Ros; apoptosis/necrosis was evaluated using AnnexinV and DAPI. Flow cytometry data was obtained using the LSR Fortessa flow cytometer.
FIG. 10(a) is representative dot plots (n=1) showing mRAGE positive and negative sperm populations with corresponding DAPI, MitoTracker and Annexin V-stained sperm sub-populations.FIG. 10(b) is evaluation of cell permeability (DAPI positive cells), and MMP (cells with Low MMP) in RAGE positive and negative sperm populations (n=35; % Mean±SEM). Paired t-test; ****=P<0.0001.FIG. 10(c) is evaluation of apoptotic, early apoptotic and necrotic sperm cells in RAGE positive and negative sperm populations (n=35; % Mean±SEM). Paired t-test; ****=P<0.0001. - There was a clear distinction between cell permeability, MMP and apoptosis and necrosis in mRAGE positive and negative cell populations when analysing the flow cytometry dot plots (
FIG. 10(a) ). Statistical analysis revealed a 3-fold increase in DAPI staining, representing non-viable cells was found in mRAGE positive cell population (FIG. 10(b) , P<0.0001) compared to mRAGE negative cell populations. A 10-fold increase in cells with high MMP was found in mRAGE negative cell populations (FIG. 10(b) , P<0.0001) compared to mRAGE positive cell populations. A 6- and 2.5-fold increase in apoptotic and necrotic cells, respectively, were found in mRAGE positive cell populations compared to mRAGE negative cell populations (FIG. 10(c) , P<0.0001). - The next set of experiments were set up to determine if mRAGE protein expression differed following separation of highly motile sperm from the unprocessed semen sample. Highly motile progressive sperm can be isolated from the total sperm population using the swim-up purification method and/or from DGC purification.
-
FIG. 11(a) shows representative dot plots (n=1) showing mRAGE positive and negative sperm populations in unprocessed (Neat) and in sperm processed by DGC and Swim-Up.FIG. 11(b) shows evaluation of sperm cells expressing mRAGE (mRAGE positive sperm cells) in unprocessed and processed sperm via DGC and Swim-Up (n=15; % Mean±SEM). Corresponding unprocessed (neat) fractions for each processing methods included. Paired one-way ANOVA; ****=P<0.0001.FIG. 11(c) shows evaluation of progressive motility in unprocessed and processed sperm via DGC and Swim-Up (n=15; % Mean±SEM). Corresponding unprocessed (neat) fractions for each processing methods included. Paired one-way ANOVA; ****=P<0.0001.FIG. 11(d) shows evaluation of cell permeability (DAPI positive cells), cells with High MMP and Apoptotic Sperm in unprocessed and processed sperm via DGC and Swim-Up (n=15; % Mean±SEM). Corresponding unprocessed (neat) fractions for each processing methods included. Paired one-way ANOVA; ****=P<0.0001. - A 5-fold and 4-fold decrease in RAGE protein expression (
FIG. 11(b) ; P<0.0001) was observed in the DGC and Swim-Up purified sperm. In parallel, a 3.5- and 3.4-fold increase in progressively motile sperm (FIG. 11(c) ; P<0.0001) in DGC and Swim-Up purified sperm. Alongside this, an average 2.75-fold decrease in impaired cell permeability, 2.75-fold decrease in apoptotic sperm and 1.5-fold increase in MMP (FIG. 11(d) ; P<0.0001) was observed in the DGC and swim-up purified sperm. - In summary, sperm purified using both methods discussed here result in highly motile progressive, live sperm that have impermeable cell membranes and highly active mitochondria. In addition, the decrease in mRAGE protein expression in the purified fractions would suggest that mRAGE expressing sperm are removed during the purification process.
-
FIG. 12(a) shows evaluation of the correlation between mRAGE expressing sperm (mRAGE positive cells) and immotile cells present after DGC and Swim-Up processing methods (n=15% Mean±SEM).FIG. 12(b) shows evaluation of the correlation between sperm not expressing mRAGE (mRAGE negative cells) and motile sperm present after DGC and Swim-Up processing methods (n=15).FIG. 12(c) shows evaluation of the correlation between sperm not expressing RAGE (RAGE negative cells) and progressive sperm present after DGC and Swim-Up processing methods (n=15). Pearson's correlation R2 value indicated on graph; error bars within 95% CI shown by dashed line. ****=P<0.0001. - Further analysis of the data, whereby correlation plots were generated analysing mRAGE protein expression and sperm motility of processed samples, revealed statistically significant correlations between mRAGE protein expression and sperm motility. The statistically significant correlations were observed between mRAGE positive cells and immotile sperm (
FIG. 12(a) ; R2>0.84), mRAGE negative cells and motile sperm (FIG. 12(b) ; R2>0.82) and mRAGE negative cells and progressively motile sperm (FIG. 12(c) ; R2>0.39). - This data confirms that both Swim-Up and DGC purification methods result in a highly progressive sperm fraction with minimal mRAGE expressing sperm. Any residual mRAGE expressing sperm within the purified fractions could be associated with the small percentages of static/slow sperm remaining after the processing event.
- In conclusion, the results from these experiments indicate that mRAGE protein expression correlates with static sperm, whereas conversely, sperm cells not expressing mRAGE are highly motile healthy sperm.
- Evaluation of two methods of sperm motility assessments—CASA and manual motility assessment and the correlation with normal VS abnormal morphology in unprocessed semen samples. Sperm morphology was evaluated using Test-Simplet slides and strict Kruger criteria. CASA motility assessment was carried out using the Hamilton Thorne CASA software. Sperm manual motility assessment and morphology assessment was carried out at Merrion Fertility Clinic as part of their routine semen analysis procedures.
FIG. 13(a) Evaluation of sperm motility (static sperm, motile sperm, progressive sperm) measured via CASA in patients with normal and abnormal morphology (n=35; Mean SEM). Unpaired t-test; ***=P<0.0005; **=P<0.005.FIG. 13(b) Evaluation of sperm motility (static sperm, motile sperm, progressive sperm) measured via manual motility assessment in patients with normal and abnormal morphology (n=35; Mean SEM). Unpaired t-test; ***=P<0.0005. - A statistically significant increase in static motility measured using CASA (
FIG. 13(a) ) and manual methods (FIG. 13(b) ) was observed in sperm samples with abnormal morphology (P=0.0048 and P=0.0007, respectively). Parallel results were observed when comparing total motility and progressive motility with sperm morphology (FIG. 13(a) andFIG. 13(b) ; P values ranging from 0.0030-0.007). There is a difference in the statistical significance between the two motility assessment methods, however the trend between the two groups is similar. This data proves the already known phenomenon that motility and morphology correlate with one another and are indicators of sperm health. -
FIG. 14(a) shows the evaluation of RAGE protein expression, cell permeability (DAPI positive cells), and MMP (cells with low MMP) in patients with normal and abnormal morphology (n=35; % Mean±SEM). Unpaired t-test; ***=P<0.0005. *=P<0.05.FIG. 14(b) shows the evaluation of apoptotic, early apoptotic and necrotic sperm cells in patients with normal and abnormal morphology (n=35; % Mean±SEM). - Subsequent analysis of the flow cytometry data and morphology data, carried out using paired t-tests, revealed a statistically significant difference between RAGE protein expression and MMP (
FIG. 14(a) ; P=0.0002 and P=0.0236, respectively) in patients with normal and abnormal morphology. This data complements the previously determined data and further confirms our hypothesis that mRAGE protein expression is associated with unhealthy, static sperm and now revealing a new relationship with morphologically abnormal sperm. - Table. 1 shows neat samples from processed vs unprocessed experiments (N=15). Mean Fluorescent Intensity (MFI; geometric mean) was analysed in sperm cells that are mRAGE positive vs mRAGE negative, and MFI was analysed in sperm cells with High MMP vs low MMP.
- Table. 1 shows the percentage of sperm cells that are mRAGE and mRAGE. Also shown is the MFI (geometric mean) of the sperm cells that are mRAGE positive and mRAGE negative. Also shown is the fold difference between the MFI of mRAGE positive vs mRAGE negative sperm cells. The average fold difference in MFI between mRAGE positive vs mRAGE negative sperm cells is 21.1% +/−13.8%
- Table. 1 shows the percentage of sperm cells with high MMP and low MMP. Also shown is the MFI (geometric mean) of the sperm cells that have high MMP vs low MMP. Also shown s the fold difference between the MFI of sperm cells with high MMP and low MMP. The average fold difference in MFI between cells with high vs low MMP is 25.3% +/−10.1%.
-
TABLE 1 Analysis of quantifying mRAGE positive and mRAGE negative sperm cells, and Mitochondrial Membrane Potential (MMP) MMP fluorophore - 540 RAGE fluorophore - 640 MFI (geometric mean) MFI (genometic mean) Sample Sample Percentage sperm cells Fold Percentage sperm cells Fold # ID mRAGE+ mRAGE− mRAGE+ mRAGE− difference High MMP Low MMP High MMP Low MMP difference 1 612 18.4 81.2 3212 314 10.22 49.9 49.6 48223 1337 36.06 2 620 13.4 86.2 4832 282 17.13 71.6 27.1 30876 1233 25.04 3 621 16.4 83.1 5144 231 22.26 71.6 25.4 26101 1320 19.77 4 623 25 72.8 4450 220 20.22 54 43.3 33965 1568 21.66 5 624 16 82.8 4195 214 19.60 56.4 40.7 25131 999 25.15 6 625 17.5 81.7 4436 309 14.35 45.5 52.6 31103 1100 28.27 7 626 43.9 54.9 3891 277 14.04 17.1 79.1 24173 1215 19.89 8 634 22 77.4 4491 313 14.34 62.3 36.4 49928 3533 14.13 9 636 25.1 74.6 5055 332 15.22 45.2 53.1 75651 3697 20.46 10 637 27.4 72.2 6189 309 20.02 57.5 41.1 41706 1627 25.63 11 658 37.6 61.4 3271 379 8.63 50 49.4 43358 893 48.55 12 662 59.7 39.6 3068 318 9.64 14.5 81 20901 518 40.34 13 665 3.96 95.9 2991 62.4 47.93 69.7 29.4 84127 5909 14.23 14 667 3.93 95.7 1335 53 25.18 45.5 53.8 36582 1740 21.02 15 669 3.55 96.2 1951 34 57.38 61.9 37 32554 1671 19.48 Average 22.25 77.04 3900 243 21.08 51.51 46.6 40291 1890 25.31 STDEV 15.48 15.69 1275 109 13.76 17.11 16.44 18308 1418 10.11
Claims (20)
1. An in vitro method of evaluating sperm health; the method comprising the steps of:
(a) providing a biological sample from a patient;
(b) measuring the quantitative or qualitative level of a biomarker in the sample; and
(c) evaluating sperm health based on the quantitative or qualitative level of the biomarker,
wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).
2. The method according to claim 1 , wherein the evaluating step (c) comprises evaluating the presence or absence of quantifiable levels of mRAGE on sperm cells from the sample to define the patient as mRAGE negative or mRAGE positive.
3. The method according to claim 2 wherein mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm, poor sperm motility and infertility; and mRAGE negative is indicative of one or more of normal sperm and good sperm health.
4. The method according to claim 3 wherein the presence of quantifiable levels of mRAGE on sperm cells from the sample defines the patient as mRAGE positive and wherein the absence of quantifiable levels of mRAGE on sperm cells from the sample defines the patient as mRAGE negative.
5. The method according to claim 1 wherein the evaluating step (c) comprises comparing the amount of sperm expressing quantifiable levels of mRAGE to the amount of sperm not expressing quantifiable levels of mRAGE.
6. The method according to claim 5 wherein the amount of sperm expressing quantifiable levels of mRAGE deviates from the amount of sperm not expressing quantifiable levels of mRAGE by at least 1-fold.
7. The method according claim 6 , wherein deviation of the amount of sperm expressing quantifiable levels of mRAGE from the amount of sperm not expressing quantifiable levels of mRAGE by at least 1-fold defines the patient as mRAGE positive;
and/or wherein deviation of the amount of sperm not expressing quantifiable levels of mRAGE from the amount of sperm expressing quantifiable levels of mRAGE by at least 1-fold defines the patient as mRAGE negative.
8. The method according to claim 6 wherein a Mean Fluorescent Intensity value of the sperm expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value of the sperm not expressing quantifiable levels of mRAGE by at least 10-fold defines the patient as mRAGE positive; and/or wherein a Mean Fluorescent Intensity value of the sperm not expressing quantifiable levels of mRAGE deviates from a Mean Fluorescent Intensity value of the sperm expressing quantifiable levels of mRAGE by at least 10-fold defines the patient as mRAGE negative.
9. The method according to claim 1 wherein the method of evaluating sperm health further comprises a step of assessing sperm motility as IM, NP or PR as defined by the World Health Organisation (WHO) laboratory manual for the examination and processing of human semen (fifth edition, 2010), wherein sperm categorised as IM is indicative of poor sperm health; wherein sperm categorised as NP and/or wherein sperm categorised as PR is indicative of good sperm health.
10. The method according to claim 9 wherein the amount of mRAGE positive sperm is inversely correlated to the amount of PR sperm.
11. The method according to claim 1 wherein the method of evaluating sperm health further comprises a step of assessing sperm morphology wherein sperm are classified as normal when the sperm head is smooth, regularly contoured and substantially oval in shape; and sperm are classified as abnormal when there is deviation from normal, wherein deviation from normal is indicative of poor sperm health.
12. The method according to claim 1 wherein the method of evaluating sperm health further comprises a step of assessing mitochondrial membrane potential (MMP), wherein quantifiable levels of MMP is indicative of mRAGE negative sperm and the absence of quantifiable levels of MMP is indicative of mRAGE positive sperm.
13. The method according to claim 1 wherein the method of evaluating sperm health further comprises a step of assessing cell permeability, wherein there is at least a 2-fold decrease in cell permeability in mRAGE positive sperm compared to mRAGE negative sperm is indicative of poor sperm health;
and/or wherein the method of evaluating sperm health further comprises a step of assessing apoptosis and necrosis, wherein there is at least a 2-fold increase in apoptosis and necrosis in mRAGE positive sperm compared to mRAGE negative sperm is indicative of poor sperm health; and/or wherein the method of evaluating sperm health further comprises a step of assessing DNA fragmentation, wherein there is at least a 2-fold increase in DNA fragmentation in mRAGE positive sperm compared to mRAGE negative sperm, which is indicative of poor sperm health.
14. The method according to claim 1 wherein the method of evaluating sperm health further comprises a step of processing the sample using separation techniques, including any one or more of density gradient centrifugation (DGC) and the sperm swim-up method.
15. The method according to claim 14 wherein the DGC method and the swim-up method results in:
(a) at least a 2-fold decrease in mRAGE positive sperm compared to mRAGE negative sperm; and/or
(b) at least a 2-fold increase in PR sperm, and/or
(c) sperm with at least a 2-fold decrease in cell permeability, and/or
(d) at least a 2-fold decrease in apoptotic sperm;
which is indicative of sperm that can be used in assisted reproductive technologies (ART).
16. The method according to claim 1 wherein the patient is any one or more of non-diabetic, non-smoking and non-obese.
17. The method according to claim 1 wherein the method is a method of diagnosing infertility.
18. A method for treating infertility in a patient which comprises:
(a) evaluating sperm health of the patient by the method of claim 1 wherein the evaluating step (c) comprises evaluating the presence or absence of quantifiable levels of mRAGE on sperm cells from the sample to define the patient as mRAGE negative or mRAGE positive; and wherein mRAGE positive is indicative of one or more of poor sperm health, abnormal sperm, poor sperm motility and infertility; and mRAGE negative is indicative of one or more of normal sperm and good sperm health; and
(b) if the patient is defined as mRAGE positive, treating the patient for infertility.
19. A method for carrying out an assisted reproductivity technologies (ART) procedure using sperm of a patient which comprises:
(a) evaluating sperm health of the patient by the method of claim 15 and determining whether or not the sperm of the patient are useful in the ART procedure; and
(b) if the sperm of the patient are determined to be useful in the ART procedure, using the sperm of the patient to conduct the ART procedure.
20. The method of claim 19 wherein the ART procedure is any one or more of intrauterine insemination (IUI), intracytoplasmic injection (ICSI) and in vitro fertilisation (IVF).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB2014788.0A GB2598938A (en) | 2020-09-18 | 2020-09-18 | A method of evaluating sperm health and identifying sperm to be used in assisted reproductive technologies |
| GB2014788.0 | 2020-09-18 | ||
| PCT/EP2021/075820 WO2022058592A1 (en) | 2020-09-18 | 2021-09-20 | A method of evaluating sperm health and identifying sperm to be used in assisted reproductive technologies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US20240012006A1 true US20240012006A1 (en) | 2024-01-11 |
Family
ID=73196661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US18/044,511 Pending US20240012006A1 (en) | 2020-09-18 | 2021-09-20 | A method of evaluating sperm health and identifying sperm to be used in assis reproductive technologies |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20240012006A1 (en) |
| EP (1) | EP4214514A1 (en) |
| JP (1) | JP2023544508A (en) |
| GB (1) | GB2598938A (en) |
| WO (1) | WO2022058592A1 (en) |
-
2020
- 2020-09-18 GB GB2014788.0A patent/GB2598938A/en active Pending
-
2021
- 2021-09-20 US US18/044,511 patent/US20240012006A1/en active Pending
- 2021-09-20 EP EP21786098.0A patent/EP4214514A1/en active Pending
- 2021-09-20 JP JP2023517871A patent/JP2023544508A/en active Pending
- 2021-09-20 WO PCT/EP2021/075820 patent/WO2022058592A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022058592A1 (en) | 2022-03-24 |
| GB202014788D0 (en) | 2020-11-04 |
| GB2598938A (en) | 2022-03-23 |
| EP4214514A1 (en) | 2023-07-26 |
| JP2023544508A (en) | 2023-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dogan et al. | Sperm protamine-status correlates to the fertility of breeding bulls | |
| Kennedy et al. | Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination | |
| Said et al. | Effects of advanced selection methods on sperm quality and ART outcome: a systematic review | |
| Gillan et al. | Flow cytometric evaluation of sperm parameters in relation to fertility potential | |
| Garrett et al. | Clinical application of sperm-oocyte interaction tests in in vitro fertilization–embryo transfer and intracytoplasmic sperm injection programs | |
| Sellami et al. | Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility | |
| Marchetti et al. | Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome | |
| Skowronek et al. | DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men | |
| Seppo et al. | Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies | |
| Marchetti et al. | Staining of human sperm with fluorochrome‐labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters | |
| Manochantr et al. | Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men | |
| Varum et al. | Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers | |
| Liu et al. | Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm–zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction | |
| Egeberg Palme et al. | Viable acrosome-intact human spermatozoa in the ejaculate as a marker of semen quality and fertility status | |
| WO2011137906A1 (en) | Test for male fertility | |
| Mehta et al. | Identification and preparation of sperm for ART | |
| Enoiu et al. | Expression of membrane fusion proteins in spermatozoa and total fertilisation failure during in vitro fertilisation | |
| Buckman et al. | Semen levels of spermatid-specific thioredoxin-3 correlate with pregnancy rates in ART couples | |
| Liu et al. | Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm | |
| Heidari et al. | Fibronectin as a new biomarker for human sperm selection in assisted reproductive technology | |
| US20240012006A1 (en) | A method of evaluating sperm health and identifying sperm to be used in assis reproductive technologies | |
| Han et al. | Development of an optimal protocol for isolation and purification of human granulosa cells in patients with different ovarian reserves | |
| Pandya et al. | TLR4-endothelin axis controls syncytiotrophoblast motility and confers fetal protection in placental malaria | |
| US20200326331A1 (en) | Methods and compositions for determining the presence or absence of dna aberrations | |
| Garrido et al. | Flow cytometry in human reproductive biology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |